Germ line loss-of-function heterozygous mutations in the RUNX1 gene cause familial platelet disorder with associated myeloid malignancies (FPDMM) characterized by thrombocytopenia and a life-long risk of hematological malignancies. Although gene therapies are being considered as promising therapeutic options, current preclinical models do not recapitulate the human phenotype and are unable to elucidate the relative fitness of mutation-corrected and RUNX1-heterozygous mutant hematopoietic stem and progenitor cells (HSPCs) in vivo long term. We generated a rhesus macaque with an FPDMM competitive repopulation model using CRISPR/Cas9 nonhomologous end joining editing in the RUNX1 gene and the AAVS1 safe-harbor control locus. We transplanted mixed populations of edited autologous HSPCs and tracked mutated allele frequencies in blood cells. In both animals, RUNX1-edited cells expanded over time compared with AAVS1-edited cells. Platelet counts remained below the normal range in the long term. Bone marrows developed megakaryocytic dysplasia similar to human FPDMM, and CD34+ HSPCs showed impaired in vitro megakaryocytic differentiation, with a striking defect in polyploidization. In conclusion, the lack of a competitive advantage for wildtype or control-edited HSPCs over RUNX1 heterozygous-mutated HSPCs long term in our preclinical model suggests that gene correction approaches for FPDMM will be challenging, particularly to reverse myelodysplastic syndrome/ acute myeloid leukemia predisposition and thrombopoietic defects.

Germ line DDX41 variants have been implicated in late-onset myeloid neoplasms (MNs). Despite an increasing number of publications, many important features of DDX41-mutated MNs remain to be elucidated. Here we performed a comprehensive characterization of DDX41-mutated MNs, enrolling a total of 346 patients with DDX41 pathogenic/likely-pathogenic (P/LP) germ line variants and/or somatic mutations from 9082 MN patients, together with 525 first-degree relatives of DDX41-mutated and wild-type (WT) patients. P/LP DDX41 germ line variants explained ∼80% of known germ line predisposition to MNs in adults. These risk variants were 10-fold more enriched in Japanese MN cases (n = 4461) compared with the general population of Japan (n = 20 238). This enrichment of DDX41 risk alleles was much more prominent in male than female (20.7 vs 5.0). P/LP DDX41 variants conferred a large risk of developing MNs, which was negligible until 40 years of age but rapidly increased to 49% by 90 years of age. Patients with myelodysplastic syndromes (MDS) along with a DDX41-mutation rapidly progressed to acute myeloid leukemia (AML), which was however, confined to those having truncating variants. Comutation patterns at diagnosis and at progression to AML were substantially different between DDX41-mutated and WT cases, in which none of the comutations affected clinical outcomes. Even TP53 mutations made no exceptions and their dismal effect, including multihit allelic status, on survival was almost completely mitigated by the presence of DDX41 mutations. Finally, outcomes were not affected by the conventional risk stratifications including the revised/molecular International Prognostic Scoring System. Our findings establish that MDS with DDX41-mutation defines a unique subtype of MNs that is distinct from other MNs.

Treatment paradigms for B-cell non-Hodgkin lymphomas (B-NHL) have shifted dramatically in the last 2 decades following the introduction of highly active immunotherapies such as rituximab. Since then, the field has continued to witness tremendous progress with the introduction of newer, more potent immunotherapeutics, including chimeric antigen receptor T-cell therapy, which have received regulatory approval for and currently play a significant role in the treatment of these diseases. Bispecific antibodies (BsAb) are a novel class of off-the-shelf T-cell redirecting drugs and are among the most promising immunotherapeutics for lymphoma today. BsAb may target various cell-surface antigens and exist in different formats. Anti-CD20xCD3 BsAb have demonstrated remarkable single-agent activity in patients with heavily pretreated B-NHL with a manageable toxicity profile dominated by T-cell overactivation syndromes. Much work remains to be done to define the optimal setting in which to deploy these drugs for B-NHL treatment, their ideal combination partners, strategies to minimize toxicity, and, perhaps most importantly, pharmacodynamic biomarkers of response and resistance. In this review, we provide an update on BsAb development in B-NHL, from discovery to clinical applications, highlighting the achievements, limitations, and future directions of the field.

Primary myelofibrosis (PMF) is a myeloproliferative neoplasm characterized by the clonal expansion of myeloid cells, notably megakaryocytes (MKs), and an aberrant cytokine production leading to bone marrow (BM) fibrosis and insufficiency. Current treatment options are limited. TGF-β1, a profibrotic and immunosuppressive cytokine, is involved in PMF pathogenesis. While all cell types secrete inactive, latent TGF-β1, only a few activate the cytokine via cell type-specific mechanisms. The cellular source of the active TGF-β1 implicated in PMF is not known. Transmembrane protein GARP binds and activates latent TGF-β1 on the surface of regulatory T lymphocytes (Tregs) and MKs or platelets. Here, we found an increased expression of GARP in the BM and spleen of mice with PMF and tested the therapeutic potential of a monoclonal antibody (mAb) that blocks TGF-β1 activation by GARP-expressing cells. GARP:TGF-β1 blockade reduced not only fibrosis but also the clonal expansion of transformed cells. Using mice carrying a genetic deletion of Garp in either Tregs or MKs, we found that the therapeutic effects of GARP:TGF-β1 blockade in PMF imply targeting GARP on Tregs. These therapeutic effects, accompanied by increased IFN-γ signals in the spleen, were lost upon CD8 T-cell depletion. Our results suggest that the selective blockade of TGF-β1 activation by GARP-expressing Tregs increases a CD8 T-cell-mediated immune reaction that limits transformed cell expansion, providing a novel approach that could be tested to treat patients with myeloproliferative neoplasms.

T-cell acute lymphoblastic leukemia (T-ALL) is a T-cell malignancy characterized by cell subsets and enriched with leukemia-initiating cells (LICs). β-Catenin modulates LIC activity in T-ALL. However, its role in maintaining established leukemia stem cells remains largely unknown. To identify functionally relevant protein interactions of β-catenin in T-ALL, we performed coimmunoprecipitation followed by liquid chromatography-mass spectrometry. Here, we report that a noncanonical functional interaction of β-catenin with the Forkhead box O3 (FOXO3) transcription factor positively regulates LIC-related genes, including the cyclin-dependent kinase 4, which is a crucial modulator of cell cycle and tumor maintenance. We also confirm the relevance of these findings using stably integrated fluorescent reporters of β-catenin and FOXO3 activity in patient-derived xenografts, which identify minor subpopulations with enriched LIC activity. In addition, gene expression data at the single-cell level of leukemic cells of primary patients at the time of diagnosis and minimal residual disease (MRD) up to 30 days after the standard treatments reveal that the expression of β-catenin- and FOXO3-dependent genes is present in the CD82+CD117+ cell fraction, which is substantially enriched with LICs in MRD as well as in early T-cell precursor ALL. These findings highlight key functional roles for β-catenin and FOXO3 and suggest novel therapeutic strategies to eradicate aggressive cell subsets in T-ALL.

Sickle cell disease (SCD) is an inherited disorder resulting from a β-globin gene mutation, and SCD patients experience erythrocyte sickling, vaso-occlusive episodes (VOE), and progressive organ damage. Chronic hemolysis, inflammation, and repeated red blood cell transfusions in SCD can disrupt iron homeostasis. Patients who receive multiple blood transfusions develop iron overload, and another subpopulation of SCD patients manifest iron deficiency. To elucidate connections between dietary iron, the microbiome, and SCD pathogenesis, we treated SCD mice with an iron-restricted diet (IRD). IRD treatment reduced iron availability and hemolysis, decreased acute VOE, and ameliorated chronic organ damage in SCD mice. Our results extend previous studies indicating that the gut microbiota regulate disease in SCD mice. IRD alters microbiota load and improves gut integrity, together preventing crosstalk between the gut microbiome and inflammatory factors such as aged neutrophils, dampening VOE, and organ damage. These findings provide strong evidence for the therapeutic potential of manipulating iron homeostasis and the gut microbiome to ameliorate SCD pathophysiology. Many treatments, which are under development, focus on lowering the systemic iron concentration to relieve disease complications, and our data suggest that iron-induced changes in microbiota load and gut integrity are related- and novel-therapeutic targets.

Severe anemia is commonly treated with red blood cell transfusion. Clinical trials have demonstrated that a restrictive transfusion strategy of 7 to 8 g/dL is as safe as a liberal transfusion strategy of 9 to 10 g/dL in many clinical settings. Evidence is lacking for subgroups of patients, including those with preexisting coronary artery disease, acute myocardial infarction, congestive heart failure, and myelodysplastic neoplasms. We present 3 clinical vignettes that highlight the clinical challenges in caring for patients with coronary artery disease with gastrointestinal bleeding, congestive heart failure, or myelodysplastic neoplasms. We emphasize that transfusion practice should be guided by patient symptoms and preferences in conjunction with the patient's hemoglobin concentration. Along with the transfusion decision, evaluation and management of the etiology of the anemia is essential. Iron-restricted erythropoiesis is a common cause of anemia severe enough to be considered for red blood cell transfusion but diagnosis and management of absolute iron deficiency anemia, the anemia of inflammation with functional iron deficiency, or their combination may be problematic. Intravenous iron therapy is generally the treatment of choice for absolute iron deficiency in patients with complex medical disorders, with or without coexisting functional iron deficiency.

Molecular heterogeneity of diffuse large B-cell lymphoma (DLBCL) underlies the variable outcomes achieved with immunochemotherapy. However, outcomes of gene expression profiling (GEP)-defined molecular subgroups in a real-world DLBCL population remain unknown. Here we examined the prevalence and outcomes of molecular subgroups in an unselected population of 1149 patients with de novo DLBCL in British Columbia, Canada. Evaluable biopsies were profiled by fluorescence in situ hybridization (FISH), immunohistochemistry, and digital GEP to assign cell-of-origin and the so-called "double-hit signature" (DHITsig)-a signature originally described as being characteristic for high-grade B-cell lymphoma with MYC and BCL2 rearrangements (HGBCL-DH-BCL2). DHITsig was expressed in 21% of 431 germinal center B-cell-like (GCB)-DLBCL and all 55 Burkitt lymphomas examined. Reflecting this latter finding, DHITsig has been renamed the "dark zone signature" (DZsig). DZsigpos-DLBCL, non-DZsigpos GCB-DLBCL and activated B-cell-like (ABC)-DLBCL were associated with a 2 year overall survival of 57%, 89%, and 71%, respectively. 62% of DZsigpos tumors were negative for HGBCL-DH-BCL2 by FISH, but were associated with outcomes similar to HGBCL-DH-BCL2. A small group of HGBCL-DH-BCL2 that lacked DZsig expression had different molecular features compared with DZsig-expressing HGBCL-DH-BCL2 and were associated with favorable outcomes comparable to DLBCL, not otherwise specified. DZsigpos and ABC-DLBCL had a shorter diagnosis-to-treatment interval (DTI) than GCB-DLBCL, with this metric being associated with outcome. In conclusion, DZsig expression extends beyond HGBCL-DH-BCL2 and captures a poor-prognosis DLBCL subgroup with short DTI, including patients unidentifiable by routine FISH testing, that should be considered for treatment intensification or novel therapies in prospective trials.

Neutrophilia and neutropenia commonly lead to inpatient hematology consultation. Quantitative neutrophil abnormalities have a broad differential and include diagnoses that are important to recognize because they may be associated with increased mortality. Neutrophilia can reflect etiologies such as infection, medications, inflammation, splenectomy, and congenital disorders. Neutropenia can arise from infection, medications, autoimmune destruction, sequestration, nutritional deficiency, malignancy, and congenital neutropenia syndromes. In the evaluation of all abnormalities of neutrophil number, the timing of the change, and the patient's historical neutrophil count are crucial.

Patients with hypomorphic mutations in the RAG1 or RAG2 gene present with either Omenn syndrome or atypical combined immunodeficiency with a wide phenotypic range. Hematopoietic stem cell transplantation (HSCT) is potentially curative, but data are scarce. We report on a worldwide cohort of 60 patients with hypomorphic RAG variants who underwent HSCT, 78% of whom experienced infections (29% active at HSCT), 72% had autoimmunity, and 18% had granulomas pretransplant. These complications are frequently associated with organ damage. Eight individuals (13%) were diagnosed by newborn screening or family history. HSCT was performed at a median of 3.4 years (range 0.3-42.9 years) from matched unrelated donors, matched sibling or matched family donors, or mismatched donors in 48%, 22%, and 30% of the patients, respectively. Grafts were T-cell depleted in 15 cases (25%). Overall survival at 1 and 4 years was 77.5% and 67.5% (median follow-up of 39 months). Infection was the main cause of death. In univariable analysis, active infection, organ damage pre-HSCT, T-cell depletion of the graft, and transplant from a mismatched family donor were predictive of worse outcome, whereas organ damage and T-cell depletion remained significant in multivariable analysis (hazard ratio [HR] = 6.01, HR = 8.46, respectively). All patients diagnosed by newborn screening or family history survived. Cumulative incidences of acute and chronic graft-versus-host disease were 35% and 22%, respectively. Cumulative incidences of new-onset autoimmunity was 15%. Immune reconstitution, particularly recovery of naïve CD4+ T cells, was faster and more robust in patients transplanted before 3.5 years of age, and without organ damage. These findings support the indication for early transplantation.

This phase 1b trial (NCT02670044) evaluated venetoclax-idasanutlin in patients with relapsed/refractory (R/R) acute myeloid leukemia (AML) ineligible for cytotoxic chemotherapy. Two-dimensional dose escalation (DE, n = 50) was performed for venetoclax daily with idasanutlin on days 1 to 5 in 28-day cycles, followed by dosing schedule optimization (n = 6) to evaluate reduced venetoclax schedules (21-/14-day dosing). Common adverse events (occurring in ≥40% of patients) included diarrhea (87.3% of patients), nausea (74.5%), vomiting (52.7%), hypokalemia (50.9%), and febrile neutropenia (45.5%). During DE, across all doses, composite complete remission (CRc; CR + CR with incomplete blood count recovery + CR with incomplete platelet count recovery) rate was 26.0% and morphologic leukemia-free state (MLFS) rate was 12%. For anticipated recommended phase 2 doses (venetoclax 600 mg + idasanutlin 150 mg; venetoclax 600 mg + idasanutlin 200 mg), the combined CRc rate was 34.3% and the MLFS rate was 14.3%. Pretreatment IDH1/2 and RUNX1 mutations were associated with higher CRc rates (50.0% and 45.0%, respectively). CRc rate in patients with TP53 mutations was 20.0%, with responses noted among those with co-occurring IDH and RUNX1 mutations. In 12 out of 36 evaluable patients, 25 emergent TP53 mutations were observed; 22 were present at baseline with low TP53 variant allele frequency (median 0.0095% [range, 0.0006-0.4]). Venetoclax-idasanutlin showed manageable safety and encouraging efficacy in unfit patients with R/R AML. IDH1/2 and RUNX1 mutations were associated with venetoclax-idasanutlin sensitivity, even in some patients with co-occurring TP53 mutations; most emergent TP53 clones were preexisting. Our findings will aid ongoing/future trials of BCL-2/MDM2 inhibitor combinations. This trial was registered at www.clinicaltrials.gov as #NCT02670044.

We designed a prospective, observational study enrolling patients presenting for treatment of acute myeloid leukemia (AML) at 13 institutions to analyze associations between hematopoietic cell transplantation (HCT) and survival, quality of life (QOL), and function in: the entire cohort, those aged ≥65 years, those with high comorbidity burden, intermediate cytogenetic risk, adverse cytogenetic risk, and first complete remission with or without measurable residual disease. Patient were assessed 8 times over 2 years. Time-dependent regression models were used. Among 692 patients that were evaluable, 46% received HCT with a 2-year survival of 58%. In unadjusted models, HCT was associated with reduced risks of mortality most of the subgroups. However, after accounting for covariates associated with increased mortality (age, comorbidity burden, disease risks, frailty, impaired QOL, depression, and impaired function), the associations between HCT and longer survival disappeared in most subgroups. Although function, social life, performance status, and depressive symptoms were better for those selected for HCT, these health advantages were lost after receiving HCT. Recipients and nonrecipients of HCT similarly ranked and expected cure as main goal of therapy, whereas physicians had greater expectations for cure than the former. Accounting for health impairments negates survival benefits from HCT for AML, suggesting that the unadjusted observed benefit is mostly owing to selection of the healthier candidates. Considering patients' overall expectations of cure but also the QOL burdens of HCT motivate the need for randomized trials to identify the best candidates for HCT. This trial was registered at www.clinicaltrials.gov as #NCT01929408.

We compared minimal/measurable residual disease (MRD) levels evaluated by routinely used real-time quantitative polymerase chain reaction (qPCR) patient-specific assays and by next-generation sequencing (NGS) approach in 780 immunoglobulin (IG) and T-cell receptor (TR) markers in 432 children with B-cell precursor acute lymphoblastic leukemia treated on the AIEOP-BFM ALL 2009 protocol. Our aim was to compare the MRD-based risk stratification at the end of induction. The results were concordant in 639 of 780 (81.9%) of these markers; 37 of 780 (4.7%) markers were detected only by NGS. In 104 of 780 (13.3%) markers positive only by qPCR, a large fraction (23/104; 22.1%) was detected also by NGS, however, owing to the presence of identical IG/TR rearrangements in unrelated samples, we classified those as nonspecific/false-positive. Risk group stratification based on the MRD results by qPCR and NGS at the end of induction was concordant in 76% of the patients; 19% of the patients would be assigned to a lower risk group by NGS, largely owing to the elimination of false-positive qPCR results, and 5% of patients would be assigned to a higher risk group by NGS. NGS MRD is highly concordant with qPCR while providing more specific results and can be an alternative in the front line of MRD evaluation in forthcoming MRD-based protocols.

STAT3 mutations, predominantly in the DNA-binding domain (DBD) and Src-homology 2 domain (SH2D), cause rare cases of immunodeficiency, malignancy, and autoimmunity. The exact mechanisms by which these mutations abrogate or enhance STAT3 function are not completely understood. Here, we examined how loss-of-function (LOF) and gain-of-function (GOF) STAT3 mutations within the DBD and SH2D affect monomer and homodimer protein stability as well as their effect on key STAT3 activation events, including recruitment to phosphotyrosine (pY) sites within peptide hormone receptors, tyrosine phosphorylation at Y705, dimerization, nuclear translocation, and DNA binding. The DBD LOF mutants showed reduced DNA binding when homodimerized, whereas the DBD GOF mutants showed increased DNA binding. DBD LOF and GOF mutants showed minimal changes in other STAT3 functions or in monomer or homodimer protein stability. However, SH2D LOF mutants demonstrated reduced conformational stability as either monomers or homodimers, leading to decreased pY-peptide recruitment, tyrosine phosphorylation, dimerization, nuclear localization, and DNA binding. In contrast, cancer-causing SH2D GOF mutants showed increased STAT3 homodimer stability, which increased their DNA binding. Of note, a small-molecule inhibitor of STAT3 that targets the tyrosine phosphopeptide-binding pocket within the STAT3 SH2D potently inhibited cell proliferation driven by STAT3 SH2D GOF mutants. These findings indicate that the stability of STAT3 protein monomer and homodimer is critical for the pathogenesis of diseases caused by SH2D LOF and GOF mutations and suggest that agents that modulate STAT3 monomer and/or homodimer protein stability may have therapeutic value in diseases caused by these mutations.

Large-scale analyses of genomic data from patients with newly diagnosed multiple myeloma (ndMM) have been undertaken, however, large-scale analysis of relapsed/refractory MM (rrMM) has not been performed. We hypothesize that somatic variants chronicle the therapeutic exposures and clonal structure of myeloma from ndMM to rrMM stages. We generated whole-genome sequencing (WGS) data from 418 tumors (386 patients) derived from 6 rrMM clinical trials and compared them with WGS from 198 unrelated patients with ndMM in a population-based case-control fashion. We identified significantly enriched events at the rrMM stage, including drivers (DUOX2, EZH2, TP53), biallelic inactivation (TP53), noncoding mutations in bona fide drivers (TP53BP1, BLM), copy number aberrations (CNAs; 1qGain, 17pLOH), and double-hit events (Amp1q-ISS3, 1qGain-17p loss-of-heterozygosity). Mutational signature analysis identified a subclonal defective mismatch repair signature enriched in rrMM and highly active in high mutation burden tumors, a likely feature of therapy-associated expanding subclones. Further analysis focused on the association of genomic aberrations enriched at different stages of resistance to immunomodulatory agent (IMiD)-based therapy. This analysis revealed that TP53, DUOX2, 1qGain, and 17p loss-of-heterozygosity increased in prevalence from ndMM to lenalidomide resistant (LENR) to pomalidomide resistant (POMR) stages, whereas enrichment of MAML3 along with immunoglobulin lambda (IGL) and MYC translocations distinguished POM from the LEN subgroup. Genomic drivers associated with rrMM are those that confer clonal selective advantage under therapeutic pressure. Their role in therapy evasion should be further evaluated in longitudinal patient samples, to confirm these associations with the evolution of clinical resistance and to identify molecular subsets of rrMM for the development of targeted therapies.

The mechanisms of coordinated changes in proteome composition and their relevance for the differentiation of neutrophil granulocytes are not well studied. Here, we discover 2 novel human genetic defects in signal recognition particle receptor alpha (SRPRA) and SRP19, constituents of the mammalian cotranslational targeting machinery, and characterize their roles in neutrophil granulocyte differentiation. We systematically study the proteome of neutrophil granulocytes from patients with variants in the SRP genes, HAX1, and ELANE, and identify global as well as specific proteome aberrations. Using in vitro differentiation of human induced pluripotent stem cells and in vivo zebrafish models, we study the effects of SRP deficiency on neutrophil granulocyte development. In a heterologous cell-based inducible protein expression system, we validate the effects conferred by SRP dysfunction for selected proteins that we identified in our proteome screen. Thus, SRP-dependent protein processing, intracellular trafficking, and homeostasis are critically important for the differentiation of neutrophil granulocytes.

αβ T cell receptor (TCRαβ) T cells modified to express chimeric antigen receptors (CAR), are now available as authorized therapies for certain B-cell malignancies. However the process of autologous harvest and generation of patient-specific products is costly, with complex logistics and infrastructure requirements. Premanufactured banks of allogeneic donor-derived CAR T cells could help widen applicability if the challenges of HLA-mismatched T-cell therapy can be addressed. Genome editing is being applied to overcome allogeneic barriers, most notably, by disrupting TCRαβ to prevent graft-versus-host disease, and multiple competing editing technologies, including CRISPR/Cas9 and base editing, have reached clinical phase testing. Improvements in accuracy and efficiency have unlocked applications for a wider range of blood malignancies, with multiplexed editing incorporated to target HLA molecules, shared antigens and checkpoint pathways. Clinical trials will help establish safety profiles and determine the durability of responses as well as the role of consolidation with allogeneic transplantation.